INTERNATIONAL JOURNAL OF LEPROSY^

Volume 52, Number 2Printed in the U.S.A.

Preliminary Evidence of Natural Resistance toMycobacterium bovis (BCG) in

Lepromatous Leprosy'Jean-Louis Stach, Georgette Delgado, Michel Strobel,

Jacques Milian, and Philippe H. Lagrange 2

One of the most characteristic features ofleprosy is the bipolar partition of patients.Some of them, classified as tuberculoid (TT),vigorously light Mycobacterium leprac, pre-venting its growth and disseminationthroughout the body. Others, known as lep-romatous (LL), do not protect themselves,allowing the bacilli to grow and invade thetissues. Since M. leprac is an obligate intra-cellular parasite living in macrophages, ithas been hypothesized that there is somekind of primary deficiency in macrophagesfrom LL patients ( 1-3). Studies in favor ofthis hypothesis have not been confirmed,and macrophages from LL patients are nowbelieved not to be deficient ( 7) according tothe usual criteria of testing macrophage ac-tivation, which still need a more accuratedefinition. On the other hand, fitting intothe Mackaness model of infectious disease,there is general agreement that in leprosyLL patients are not protected because of alack of specific immunity against M. lepraeCI. Experimental studies of infectious dis-eases produced by intracellular multiplyingmicroorganisms have shown that this sim-ple assessment, emphasizing a single role ofthe specific immune response, was incom-plete (' 9) and that overall acquired resis-tance to an infectious agent must be con-sidered as the result of two phenomena. Thefirst one is now described as the "naturalresistance" that is demonstrated during theearly phase of the infection and is experi-

' Received for publication on 16 June 1983; acceptedfor publication in revised form on 2 November 1983.

2 J.-L. Stach, M.D.; G. Delgado, B.S.; and M. Stro-bel, M.D., Laboratoire d'Immunologte Cellulaire, In-stitut Pasteur de Dakar, Dakar, Senegal. J. Nlillan, M.D.,Institut de Leprologie, Dakar, Senegal. P. H. Lagrange,M.D., Laboratoire d'Immunobiologie du 13CG, De-partement de Physiopathologie Experimentale, InstitutPasteur, 25 rue du Docteur Roux, 75724 Paris, France.Reprint requests to Dr. Lagrange.

mentally evoked after an intravenous (IV)injection ofa low dose of pathogens CI. Thesecond one involves the specific "immuneresistance" which occurs later and is exper-imentally demonstrated by the immunegranuloma following local injections (' 9).Natural resistance to a given parasite is ge-netically controlled and is often nonspecificregarding the infective agent ( 20). For in-stance, in mice natural resistance to M.bovis, strain BCG, is governed by a single,autosomal, dominant gene located on chro-mosome 1 ( 10). This gene, named Bcg, isclosely linked or probably identical to gene(s)controlling natural resistance to two unre-lated microorganisms, namely Leishmaniadonovani (Lsh gene) and Salmonella tjphi-nzurium (Ity gene) ( 20).

More recently, the Bcg gene has beenshown also to control resistance to M. le-praemurium, the causative agent of murineleprosy (4.21 ) . Experimental evidence showedthat resident peritoneal macrophages wereable to express the natural resistance to BCGas tested by in vivo as well as by in vitrotests (22, 23 ).) During these studies, in vitromacrophage inhibition of BCG 3H uracilincorporation appeared to be an accuratetest for the Bcg gene ( 23 ). This new opportunity will lead to further studies on phe-notypic expression mechanisms of the Beggene in mice ( 22 ) and might allow feasibleexplorations of the natural resistance to my-cobacterial infections in humans, whichotherwise would have been difficult for manyobvious reasons. Furthermore, since natu-ral resistance to M. lepraemurium has beenshown to be controlled by the Beg gene inmice ( 23), it was worthwhile to evaluate ifnatural resistance to BCG in humans mightbe linked to resistance or susceptibility to

Ieprae in leprosy patients. This prompt-ed us to undertake preliminary comparative

140

52, 2^Stack et al.: Resistance to BCG in LL^ 141

studies on macrophage-induced inhibitionof 3 H uracil uptake into BCG in leproma-tous and tuberculoid patients, using a testdescribed by Rook and Rainbow (' 8). Thepresent report shows somehow unexpectedresults: LL patients' blood monocytes inculture being more "naturally resistant" toBCG than monocytes from TT patients.

MATERIALS AND METHODSPatients. All tested patients were from the

Institut de Lêprologie in Dakar (Dr. Millan),and were typed according to the clinical,bacteriological, immunological, and histo-logical criteria of the Ridley and Joplingclassification (''). Their ages varied from 18to 60 years. They have been studied andcoded by pairs (one LL and one TT). Theircells were treated strictly in the same waythroughout the study. To avoid any even-tual role of chemotherapeutic agents, onlynewly detected patients, free of any treat-ment, were included in this study.

M. bovis, strain bacille Calmette-Guerin(BCG). The BCG used was the Pasteur strain(1173P2) regularly grown in liquid Sautonmedium at the Institut Pasteur de Dakar inorder to produce commercially availablevaccine. Six- to ten-day culture tubes wereshaken and centrifuged at 1500 rpm for 5min to get rid of large clumps. The singleorganism suspension was adjusted to thedesired concentration in cell culture medi-um after determination of the number ofbacilli per ml in an appropriate countingchamber.

Collection of macrophages. Monocyteswere obtained from 20 ml of blood by veni-puncture taken on heparin (10 ILJ/m1) (Li-quemine, Roche Lab.). Mononuclear cellswere isolated on a Ficoll-Hypaque (Phar-macia) gradient according to the usualmethodology, washed twice in Hanks' buff-ered salt solution (HBSS) and suspended at4 x 10 6 cells/ml in buffered RPM I medium1640 (Flow Laboratories) supplementedwith 2 mM L-glutamine (Gibco) and 10%pooled heat-inactivated human AB serumfrom normal subjects. The cells were al-lowed to adhere on a 35 mm plastic Petridish for 1 hr at 37°C in a 5% CO, incubator.After vigorous agitation, nonadherent cellswere discarded. This procedure was repeat-ed twice after addition of prewarmed cul-ture medium. Adherent cells, thereafter

named macrophages, were removed after a30 min treatment with EDTA added to themedium (3 mg/ml). As judged by May-Griinwald Giemsa (MGG) staining, theywere 95% pure monocytes-macrophages.Recovered cells were washed 3 times withcold HBSS, and once with culture medium,then adjusted to 2 x 10' cells/ml and putinto 96 well microtiter culture plates (100pl per well) which were placed for 1 hr at37°C in a 5% CO, incubator. After this in-cubation period, monolayers were washedonce again before performing the assay. Atthis stage, control wells showed almost purepopulations of macrophages (MGG stain-ing).

Measurement of 3H uracil incorporationinto BCG. The test was performed accord-ing to Rook and Rainbow ('s) with somemodifications. Since the rate of 3 H uracilincorporation depends largely on the strainof BCG and its physiological state ( 22) andsince low numbers of BCG were used toinfect macrophages resulting in very low rateof 3 H uracil incorporation into BCG, higherinfectious ratios (numbers of BCG per mac-rophage) ranging from 3/1 to 50/1 were used.Microliter wells, as previously prepared,were washed once and the culture mediumover the cell monolayer was replaced by 100pl of the appropriate BCG suspension toachieve the infecting ratio indicated above.Two controls were included, consisting ofwells containing macrophage monolayersoverlaid with medium alone, and wells freeof macrophages but seeded with 100 pl ofthe bacilli suspensions.

Plates were incubated at 37°C in a 5% CO,incubator for 4 days. Saponin (0.1% finalconcentration) was added to each well andthe plates were incubated 1 hr further to lyremacrophages. Finally 10 pl of 3 H uracil con-taining 1 pCi (51 pCi/nmol, Amersham,TRK 408) was added and, after a further24 hr incubation, the content of each wellwas recovered with a Skatron cell harvester(wells flooded with saline, TCA 5% and puremethanol). TCA precipitable radioactivityon Whatman filters was measured in countsper minute (cpm) for 1 min in 5 ml scintil-lation liquid (Biofluor, New England Nu-clear) in a liquid scintillation counter (In-tertechnique, France).

All the determinations were made in qua-druplicate samples. No antibiotic was added

40

0

142^ International Journal of Leprosy^ 1984

0.61.2 2.5^5^

10BCG PER WELL [x10 -6 ]

THE FIGURE. In vitro effect of macrophage-BCGinteraction on 3 H uracil incorporation into bacilli.Monolayers of macrophages from tuberculoid leprosyPatient 225 (^—^) and lepromatous leprosy Patient226 (■—■) containing 2.10 5 cells/well were preparedin microliter plates as indicated in Materials and Meth-ods. Washed monolayers of cells were overlaid with100 p1 of BCG suspension (in cell culture medium)containing the indicated numbers of I3CG/well. Con-trol BCG wells (0-111) were seeded with 100 pl of thesame suspension without macrophages. Control mac-rophages contained macrophages alone. (Mean of qua-druplicate ± standard error of the mean.)

at any stage. Results obtained from differentexperiments were expressed graphically ascpm of 3 1-1 uracil uptake of the appropriateBCG suspension used. Since it was alwaysfound that cpm were a function of the BCGnumbers added per well, it was possible todraw regression lines. Therefore, results ofall experiments were expressed in the fol-lowing way: a regression line average cpmincorporated per well was drawn as a func-tion of the infecting ratio using the methodof least squares. The slope of that straightline was calculated for BCG alone and BCGadded to macrophages. Then, an inhibition

THE TABLE. Comparison of the inhibi-tion index of macrophages" from LL andTT patients''

LL^PATIENTS

233 226 228 221 219 214

(f),zL,

52, 2^

Stack et al.: Resistance to BCG in LL^ 143

matous). As already mentioned, results inThe Figure showed that it was possible todraw a regression line for 3 H uracil incor-poration as a function of the numbers ofBCG added per well (or infecting ratio). Themacrophage action upon BCG uracil uptakeappears as a decrease in the slope of thatstraight line. Therefore, results of all exper-iments were expressed as an inhibition in-dex of macrophages on 3 11 uracil incorpo-ration by BCG. Calculated slopes of theregression line were 739 for 13CG alone, 652for BCG plus macrophages from Patients225 (tuberculoid leprosy) and 201 withmacrophages from Patient 226 (leproma-tous leprosy). The inhibition indexes werethen 1.1 (739/652) for Patient 225 macro-phages and 3.7 for Patient 226 macro-phages. The inhibition index is then directlyproportional to the inhibitory effect of mac-rophages.

The Table shows that, in all of the ex-periments, macrophages from LL patientsalways expressed an inhibition index whichwas greater than macrophages from tuber-culoid patients. Some patients were testedtwo or three times in different combina-tions. As can be observed, the inhibitionindex value for a single LL or TT patientvaried from one experiment to another, sothe index cannot yet be considered as char-acteristic of the patient. The comparisonsbetween two persons are only accurate, inthe conditions used, if they are tested duringthe same experiment. Nevertheless, it mustbe stressed that in any of the tested com-binations of patients, LL macrophages al-ways expressed inhibition indexes greaterthan those of TT patients. Thus, this tendsto indicate that any LL macrophage is al-ways more aggressive against BCG than amacrophage from any TT subject.

DISCUSSIONHuman blood monocvtes in culture have

been shown to have a potent inhibitory ef-fect upon BCG metabolism as judged by thelevel of incorporation of 3 1-I uracil, a RNAprecursor, confirming preceding results ob-tained by Rook and Rainbow (Is). This pre-cursor was not incorporated by macro-phages in appreciable amounts under theconditions used. Therefore, 3 /1 uracil can beused as a marker of the BCG metabolismwhich appears to be depressed in the pres-

ence of macrophages. Using this test, newevidence showed that macrophages from LLpatients are not intrinsically deficient com-pared to TT macrophages. On the contrary,they appeared to be more inhibitory on BCGmetabolism than did TT macrophages.

It recently has been shown in a mousemodel that macrophage inhibition of 1 Huracil incorporation was an accurate in vitrotest of natural resistance to BCG. Non-in-duced, resident, peritoneal macrophages ex-planted from a naturally resistant, C3Hstrain of mice as defined in rho (m), weremore inhibitory for BCG uracil incor-poration than resident peritoneal macro-phages from a naturally susceptible C57BL/6strain ( 23). The experiments reported herewere not performed under these conditionsin which non-induced resident macro-phages from normal mice were used. Thiswas not done because it has been shown thathigh lysosomal activity is found in the mac-rophages of these chronically infected pa-tients ( 25 ). Additionally, the blood-derivedmacrophages used in the present study mightbe a part of a subpopulation, or cells in aphysiological state which might be very dif-ferent from the normal resident macro-phages of mice. For example, they might beimmune-activated or immune-suppressedmacrophages in the different human pop-ulations tested. It is reasonable to speculate.but not proved, that non-adherent-depletedmonolayers of macrophages used in thiswork were not contaminated with T cells.There is also evidence that isolated mac-rophages lose their immune activation statewhen they are separated from the influenceof T cells ( 6 ). Since TT patients are knownto have a strong cellular immune response,while the LL response is depressed, onewould have expected TT macrophages to beactivated and LL macrophages suppressed.Since the opposite occurred, in repeated ex-periments. it can be considered that the re-spective immune response of the patientsdid not have any influence on the results.

If we assume that, as in the mouse model,the test is an accurate in vitro test for naturalresistance, we must consider LL patients asnaturally resistant to BCG and TT patientsas susceptible. A mouse naturally resistantto BCG has on its chromosome I the Bcgrallele of the Beg gene. This allele makes themouse able to inhibit the growth of BCG

144^ International Journal of Leprosy^ 1984

and is also associated with a higher resis-tance to Al. frpraeimerium infection (4 . ' 2 . 24 ).If one transposes the results from mousestudies to humans, it can be speculated thatLL patients have an equivalent of the Beg'allele, while TT patients bear the !kg' allele.LL patients, therefore, must be naturally re-sistant to Al. leprac and TT patients, nat-urally susceptible. Obviously there is an ap-parent paradox here.

It is hard to understand how a subjectresistant to Al. lcprac can let himself be in-vaded by the bacilli. However, careful ob-servations of murine experimental datashow that the situation is not so paradoxical("). A mouse is defined as naturally resistantto BCG if there is no growth or limitedgrowth of bacilli in the spleen three weeksafter IV injection ofa small dose (10 4/mouse)of dispersed bacilli (I"). According to thisprotocol, C3H mice were classified as a nat-urally resistant strain; C57BL/6 mice beingnaturally susceptible. C3H mice were alsodescribed ( 4) as resistant (according to thesurvival time) to 1 x 10 8 M. lepraemuriuminjected IV. But if C3H mice were inocu-lated subcutaneously with 1 x 10 7 Al. lep-raemurium, they behave as susceptible, al-lowing the bacilli to grow and to spread freelyinto the tissues ('). On the contrary, C57BL/6mice injected subcutaneously with the sameinoculum of Al lepraemurium behave asresistant, limiting bacillary invasion throughthe formation of a local immune granuloma( 5 . ' 2). So according to the route, the inoc-ulum dose, and the absence or presence ofimmunopathological reactions, the samestrain of mice can be classified as either re-sistant or susceptible ("). However, this lat-ter classification of resistance or suscepti-bility is different from the former, i.e.,natural resistance or susceptibility, since itinvolved the presence or the absence of aspecific acquired immune response (").

The pattern might be the same in hu-mans, where LL would be analogous to C3Hand TT analogous to C57BL/6 mice. Thusit is necessary to consider resistance to lep-rosy as a two-step phenomenon. A first lineof defense would be the natural resistanceinvolved in the eradication of a small in-fectious inoculum, an inoculum which alonedoes not elicit an immune response or, evenif it does elicit an immune response, the

response might be involved in the inductionof tolerance. The second line of defensewould be resistance, operating through theclassical scheme: sensitization of T cellswhich recruit new cells (granuloma lbrma-tion) and activate effector cells. In spite ofcontroversial results, M. lcprac seems to re-sist the normal macrophage process of totalbacterial destruction ( 4 '''). Thus, the onlyway to limit the infectious process of dis-semination must be the formation of an im-mune granuloma (").

As described in the mouse model, C3Hmice in spite of or because of their naturalresistance do not build an accurate immuneresponse ( 12 . 14). Whether or not the failureto produce the right type of immune re-sponse is a consequence of high natural re-sistance is an intriguing possibility (''). Forinstance, one can imagine that macrophagesin charge of the natural resistance are of aspecial subpopulation of macrophages, ef-ficient in phagocytosis but weak in the an-tigen presentation function (through a lackof Ia surface antigens for instance). As shownby Gorczynski and MacRae ( 9) with Leish-mania tropica in mice, such a macrophagesubpopulation being predominant in a nat-urally resistant individual would result in apoor immune response. In this respect, thestudies by Hirschberg (") showing a failureof antigen presentation by LL macrophagesmight fit with this explanation. Howeverrecent evidence from Stoner, et al. (24)showed that blood-derived macrophagesfrom H LA-DR matched lepromatous lep-rosy patients were still able to present spe-cific Al. leprac antigens to blood-derivedlymphocytes from responder control sib-lings. This implies another mechanism forthe unresponsiveness.

The results presented in this preliminaryreport are in agreement with the results ob-tained by Yamashita, et al. ( 25), showinghigh levels of lysozyme activity in macro-phages from lepromatous leprosy nodules.It has already been shown that lysosomalenzyme levels rose when monocytes werecultured in vitro ( 8 ) but decreased when lym-phokines, such as macrophage migration in-hibitory factor (M IF), were added ( 6 ). Theseobservations support our point of view thatblood-derived macrophages in LL nodulesor LL blood monocytes in culture behave

52, 2^Stach, et al.: Resistance to BCG in LL^ 145

as naturally more active macrophages thanthose from TT patients.

The assay described in this study, with itspreliminary results, opens a new way toevaluate the natural resistance of an indi-vidual to mycobacterial infection and mightrepresent, after repeated independent stud-ies, an accurate test for the detection of high-risk, nonresponsive subjects in leprosy or intuberculosis.

SUMMARYAn assay system has been developed based

on radiometric quantification of 3 H uracilincorporation into viable BCG in the ab-sence or presence of blood monocytes incultures from untreated lepromatous (LL)or tuberculoid (TT) leprosy patients. 3 HUracil incorporation into BCG was inhib-ited when the bacilli were cultivated in thepresence of blood-derived macrophages inculture for four days, and that inhibitionwas always greater with macrophages har-vested from LL patients compared to TTpatients. The reasons for such an observeddifference in humans are discussed accord-ing to our knowledge obtained in murinemodels of mycobacterial infections.

RESUMENSe desarroll6 un metodo para medir la incorporaciOn

do uracilo- 3 11 pot bacilos BCG viables cultivados enausencia o en presencia de monocitos provinientes depacientes con lepra lepromatosa no tratada (LL) o depacientes con lepra tuberculoide (TT). La incorpora-ciOn del uracilo- 3 1-1 por el BCG fue inhibida en pre-sencia de monocitos sanguineos cultivados por 4 dias,pero la inhibici6n fue siempre mayor en presencia demacr6fagos provinientes de pacientes LL que en pre-sencia de macrOfagos derivados de pacientes TT. Sediscuten las razones de este diferente comportamientoen humanos en base a lo que se sabe de las infeccionesmicobacterianas en modelos murinos.

RESUME

On a dáveloppe un systême devaluation base sur laquantification radiometrique de l'incorporation du ra-dical uracil marque a l' 3 H par le BCG viable en absenceou en presence de monocytes sanguins provenant demalades de 1Cpre lepromateuse (LL) ou (TT), et mis enculture. L'incorporation d'uracil 3 H dans le BCG a etcinhibe lorsque les bacilles êtaient cultives en presencede macrophages derives du sang et cultives pendantquatre fours; cette inhibition etait toujours plus pro-noncee lorsqu'on utilisait des macrophages recueillis a

partir de malades LL, que quand on faisait usage demacrophages preleves chez des maladcs TT. Les rai-sons de cette difference chez l'homme sont discutees,a la lurniere des connaissances obtenues dans les mo-deles murins d'infections bacteriennes.

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